Rickettsia rickettsii is the tick-borne etiologic agent of Rocky Mountain spotted fever. R. rickettsii is the prototypic spotted fever group rickettsia. Several other species, R. conorii, R. siberica, R. japonica, R. akari, and others cause diseases of lesser severity. Still other species in the spotted fever group, R. montana, R. peacockii, R. belli, and R. rhipicephali, are considered avirulent as they have never been associated with human disease nor do they cause overt disease in standard laboratory animals. The typhus group of rickettsia, typified by R. prowazeki, the agent of epidemic typhus, include some of the historically most devastating disease agents known to mankind. The typhus group also includes species of lesser or no virulence potential to humans. R. prowazeki and R. rickettsii are classified as Biodefense Catagory B and C agents, respectively. Rickettsia rickettsii is a member of the spotted fever group rickettsiae and the etiologic agent of Rocky Mountain spotted fever (RMSF). R. rickettsii is a small obligate intracellular Gram-negative organism maintained in its tick host through transovarial transmission. Infection with R. rickettsii occurs through the bite of an infected tick. Once the organism gains access to the host it is able to replicate within the host vascular endothelial cells and spread from cell to cell by polymerizing host cell actin. Damage to vascular endothelial cells by R. rickettsii leads to increased vascular permeability and leakage of fluid into the interstices causing the characteristic rash observed in RMSF. Infection with R. rickettsii results in a severe and potentially life threatening disease if not diagnosed and treated properly. While much is known about the progression of disease, the molecular mechanisms involved in the pathogenesis of RMSF are poorly understood. Strains of Rickettsia rickettsii vary dramatically in their virulence in animal model systems and severity of human disease. The obligate intracellular lifestyle of rickettsiae and the lack of tractable genetic systems make it difficult to identify genes involved in virulence. With the completed sequences of multiple rickettsial species, it has become possible to investigate differences between virulent and avirulent strains of rickettsiae through comparative genomics. In a guinea pig model of infection, the severity of disease as assessed by fever response varies from the most virulent, Sheila Smith, less virulent Morgan and Sao Paulo, moderate virulence for the R strain, low for HLP, and Iowa which causes no fever. Plaque morphologies also vary in size and degree of host cell lysis. To identify genes involved in the virulence of R. rickettsii, the genomes of four different strains were resequenced by comparison to known genome sequences (CGS). R. rickettsii Morgan, HLP, R and Sao Paulo strains were compared to avirulent strain R. rickettsii Iowa and virulent R. rickettsii Sheila Smith. SNP analysis revealed the Montana strains Sheila Smith and R to be highly similar while the Eastern strains Iowa and Morgan were more similar to each other. Sao Paulo appears to be most like the Montana strains, whereas HLP contained too many differences to categorize. The region of ompA containing 13 tandem repeats was sequenced using the transposon system EZ-TN5 revealing only 7 shared SNPs (4 nonsynonymous) for R and Morgan strains compared to Sheila Smith with an additional 16 SNPs identified in Morgan. Analysis of rompB, another major surface antigen, shown in Iowa to have a defect in processing, reveals identical sequences between Iowa and Morgan and R being identical to Sheila Smith. By immunoblotting, both rOmpA and rOmpB appear similar among the more virulent strains, whereas Iowa and HLP do not. The number of coding sequence SNPs between sequences is low, narrowing the field of possible virulence factors. Strains of Rickettsia rickettsii, the tick-borne agent of Rocky Mountain spotted fever. vary considerably in virulence. Genomic comparisons of R. rickettsii strains have identified a relatively small number of genes divergent in an avirulent strain. Among these is one annotated as Rickettsia Ankyrin Repeat Protein 2 (RARP-2). Homologs of RARP-2 are present in all strains of R. rickettsii, but the protein in the avirulent strain Iowa contains a large internal deletion relative to the virulent Sheila Smith strain. RARP-2 is secreted in a type IV secretion system dependent manner and exposed to the host cell cytosol. RARP-2 of Sheila Smith co-localizes with multi-lamellar membranous structures bearing markers of the endoplasmic reticulum whereas the Iowa protein shows no co-localization with host cell organelles and evidence of proteolytic degradation is detected. Overexpression of Sheila Smith-RARP-2 in R. rickettsii Iowa converts this avirulent strains typically non-lytic or opaque plaque type to a lytic plaque phenotype similar to that of the virulent Sheila Smith strain. Mutation of a predicted proteolytic active site of Sheila Smith RARP-2 abolished the lytic plaque phenotype but did not eliminate association with host membrane. RARP-2 is thus a type IV secreted effector and released from the rickettsiae into the host cytosol to modulate host processes during infection. Overexpression of Sheila Smith-RARP-2 did not, however, restore the virulence of the Iowa strain in a Guinea pig model; likely due to the multifactorial nature of rickettsial virulence. .